Summary Chimeric antigen receptor-engineered T cells (CAR T cells) provide a breakthrough for personalized cancer therapy. In this approach, a gene encoding a CAR targeting tumor antigens is delivered into patient T-cells ex vivo using a lentiviral vector, then cells reinfused into patients. The engineered CAR T cells expand in the patient and attack and destroy tumor antigen-positive cancer cells. Robust clinical responses are seen with CAR T cell therapy in some leukemias such as pediatric acute lymphocytic leukemia (ALL), but lower rates of response in adult ALL, chronic lymphocytic leukemia (CLL) and multiple myeloma. One consequence of integration of a CAR- encoding lentiviral vector in patient T cells is local disruption of the host genome. We recently published an example where the resulting insertional mutagenesis bolstered successful therapy--patient T-cell expansion was associated with an integration event in TET2, which encodes an enzyme involved in CpG demethylation, and this was mechanistically linked with enhanced T cell function and durable remission. Here we take advantage of data from insertional mutagenesis of patient CAR T cells to identify genes and pathways of particular importance for effective anti-tumor activity. TGFBR2 provides a second example of where insertional mutagenesis was associated with expansion of CAR T cells, and separate studies have also implicated reduced function of this gene as associated with improved CART function. Intense efforts are now under way to modulate both of these pathways to enhance therapeutic success. We have completed longitudinal analysis of integration site distributions in 40 CAR T-treated subjects, targeting both ALL and CLL, and find numerous examples of clonal expansions in patients successfully responding to therapy, providing a unique window on CAR T cell function. We have in hand samples from another 266 subjects, some of whom are responders showing long term persistence of CAR T cells. We have further devised a series of assays in cell culture and mouse models to modulate activity of targeted genes and characterize CAR T cell proliferation and anti-tumor activity. Thus we propose to investigate these genes and pathways in detail and develop means for manipulating them clinically. We propose the following Specific Aims: Aim 1. Elucidate the rules governing superior CAR T cell proliferation and persistence taking advantage of lentiviral integration as an insertional mutagen. Aim 2. Carry out functional analyses of genes implicated in vector driving of CAR T cells to identify proteins and pathways important for CAR T proliferation, persistence and anti-tumor activity. The output of this project will be methods for manipulating genes and pathways important for effective CAR T proliferation and function, which will then be taken directly into clinical development.